Cloning:Recombinant DNA

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CloningRecombinant DNA
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Multistep Process

• . Produce fragments of DNA using enzymes that cut
  DNA at specific base sequences.
• . Link these fragments to self-replicating forms
  of DNA vectors.

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• . Replicate the recombinant DNA molecule in the
  host organism (1000s of copies).

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• . Retrieve the cloned copies for use or
  modification.
• . Produce and purify gene product.

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Some useful definitions
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Restriction Enzymes

• Enzymes that recognize a specific base sequence
  in DNA and cleave at that site
• Isolated from bacteria that inactivated viruses
  via cutting their DNA
• Molecular scissors

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Recognition sequence

• Palindrome - sequence is read the same on either
  strand, when read from 5 to 3
• Creates either sticky ends or blunt ends

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Eco R1
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Vectors

• A self-replicating DNA molecule that is used to
  transfer foreign DNA fragments between cells.

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Steps in Cloning
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Steps in cloning - General

• Isolate vector DNA and gene of interest
• Cut both with the same restriction enzyme
• Mix DNAs and ligate recombinant DNA

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• Transfer recombinant molecule into host cell
  (transform)
• Grow/Select transformants

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Types of Vectors and DNA delivery systems
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Types of Vectors

• Plasmid
• Phage (virus)
• Cosmid
• Yeast Artificial Chromosome (YAC)

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Plasmids

• Circular extrachromosomal DNA molecules naturally
  found in bacteria
• Self-replicating
• Can insert pieces up to 10kb

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Plasmid vectors need

• origin of replication
• selectable marker (antibiotic)
• unique restriction enzyme cleavage sites

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Plasmid Placement in Cell
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Phage vectors

• Derivatives of phage ? (lambda)
• Linear DNA
• Can insert up to 15 kb fragments

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Phage Insertion
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Cosmids

• Dont occur naturally
• Constructed using features of both plasmids and
  phage ?
• Can carry inserts up to 45 kb

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YACs
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YACs
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YACs

• Yeast artificial chromosome
• Self-replicating elements
• Can insert segments up to 1 million base pairs
• Can replicate any inserted DNA via transfer to
  yeast cells

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Essential elements for YACs

• Tel - telomeres
• Cen - centromere
• Ori - Origin of replication
• Selectable markers
• Restriction enzyme recognition sites

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Particle Gun

• Usually using cell culture
• Shoot DNA coated objects into cells
• Tungsten pellets, Whiskers

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We can insert the gene into cells Now what?

• Selecting for transformed cells and amplifying
  the product

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Basic Steps

• Identify the transformants
• Isolate transformed colonies
• Amplify the product

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Identifying transformants

• Vectors containing antibiotic resistance genes
  can be used
• Those that took up the vector will now express
  antibiotic resistance
• Ability to metabolize substances included in
  media

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Isolate Colonies of Interest
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Amplify the Product

• Use bacteria (usually E. Coli) to amplify product
• Sometimes yeast cells, if the gene you are
  amplifying is a eukaryote specific gene

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Genetic Libraries
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Genetic library

• Collection of clones that contains all the
  genetic information of an individual genomic
  library - gene bank
• Chromosomes, set of genes of single cell type etc.

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• cDNA - mRNA population made into cDNA. Produce
  clones

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• Can recover genes of interest from libraries for
• Clinical studies
• Evolutionary comparison
• Experimental studies
• Commercial use

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Construction of...

• DNA isolated from an organism
• Digest into smaller segments which can be
  inserted within vectors (size limitations)

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• record of genome or portion of
• Can be screened, hybridization

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